The work described herein was supported by the National Institute of Health under grant numbers K08-NS-01713 and R01-NS-24464. The government may have certain rights in the invention.
c-myb is a nuclear phosphoprotein involved in the regulation of cell proliferation and differentiation. The expression of the c-myb gene is greatest in cells as they prepare to enter and traverse the G1/S transition phase of the cell cycle. The expression of this gene subsequently decreases as cells terminally differentiate. Recent studies have shown that c-myb binds to specific DNA sequences and transactivates transcription of DNA polymerase-alpha and cdc2, two genes that are critical for DNA synthesis. (Baseraga et al., Ann. NY Acad. Sci., 660:64-69 (1992); Ku et al., J. Biol. Chem., 268:2255-2259 (1993).)
c-myb RNA levels are exceptionally high during the proliferation of immature thymocytes, prostate, and during the development of the central nervous system (CNS). Thompson et al., Nature, 319:374 (1986); Thiele et al., Mol. Cell Biol. 8:1677 (1988). More recent studies have demonstrated that c-myb expression is required for T-lymphocyte proliferation and the proliferation of intermediate-late myeloid and erythroid progenitors, but less important for early progenitor cell amplification. Caracciolo et al., J. Clin. Invest., 85:55 (1990). In human HL-60 leukemic cells, Brelvi and Studzinski reported that c-myb RNA was elevated in cell populations enriched for S-phase cells. Graser et al., J. Cell Physiol., 131:43 (1987). However, not all cells utilize c-myb protein during the proliferative process, and may use other members of the myb family (e.g., b-myb) to carry out these functions.
c-myb is a cellular homolog of the transforming gene v-myb, found in avian myeloblastosis virus (AVM) and the E26 virus. These avian viruses cause myeloblastic leukemias in chickens and can transform myelomonocytic cells in culture. Furthermore, the oncogenic activation of c-myb in chicken hematopoietic cells and murine myeloid tumor cells has been shown to be associated with 5' and 3' truncations. (Cuddihy et al., Mol. Cell. Biol., 13:3505-3513 (1993).) The human c-myb gene has been found to be altered in human glioblastoma multiforme cell lines. The degree of amplification in four cell lines was 10-fold as determined by densitometry. A rearrangement within the coding region and an enhanced gene activity were also noted. Welter, C. et al., Cancer Letters, 52:57 (1990).
Antisense oligonucleotides (AONs) are specific nucleic acid sequences that interfere with the translation of mRNA into protein. The administration of antisense to c-myb has recently been shown to inhibit the proliferation of T-cells and other hematopoietic cells. Venturelli, D. et al., PNAS, 87:5963 (1990). In studies of human myeloid leukemia cells, c-myb antisense was found to suppress the proliferation of HL-60, ML-3, KG-1, and KG-1a cell lines while antisense to c-myc or c-fes had no effect. Ferrari et al., Cell Growth Differ., 1:543 (1990).
Antisense oligonucleotide sequences complementary to DNA sequences encoding growth factors have been used in vitro to inhibit the growth of malignant brain tumor cell lines in a few instances. For example, 14-mer phosphorothioate oligodeoxynucleotides targeted against PDGF-A-chain-, -B-chain-, and -bFGF-mRNA, were used to inhibit cell proliferation of two glioblastoma cell lines, HTZ-146 and HTZ-17. Behl, C. et al., Neurosurgery, 33:674 (1993). In the A172 glioblastoma cell line, antisense oligonucleotides complementary to c-sis mRNA inhibited cell proliferation in a time- and dose-dependent fashion. The c-sis oncogene encodes the B-polypeptide chain of PDGF. Nitta, T. et al., Neurosurgery, 34:309 (1994). The addition of bFGF-specific antisense oligonucleotide to the U87 glioblastoma cell line significantly inhibited the growth rate of these cells within 48 hours and blocked proliferation beyond 2 days. Murphy et al., Molecular Endocrinology, 6:877 (1992).
One study has examined the effects of an antisense oligonucleotide to the c-myb oncogene on neuroectodermal tumor cells. Neuroectodermal cell lines give rise to neoplasms that typically originate in the peripheral nervous system and spinal cord, and are not considered to have originated in the brain. In this study, Raschella et al. found that c-myb antisense oligonucleotide down-regulated MYB protein and inhibited the proliferation of neuroectodermal cell lines. Raschella et al., Cancer Res., 52:4221 (1992).
Glioblastoma multiforme is one example of a brain tumor and is often fatal. To this date there are few effective treatments for brain tumors. Current treatment methods such as surgery, radiation therapy, and systemic chemotherapy for malignant brain tumors have not resulted in a significant prolongation in survival. The 2-year survival rate for the most common brain tumor, the glioblastoma multiforme, is less than 20% and disseminated disease in the cerebrospinal fluid has an estimated survival of 3-4 months in adults.
The lack of specificity of conventional chemotherapy for malignant cancers found in the brain has led to dose-limiting side effects and unacceptable toxicity. Because of the limitations of these conventional agents, investigators have sought new innovative treatment modalities. Immunotoxins, toxic proteins covalently linked to a tumor-specific monoclonal antibody or other carrier ligand, are one class of compounds that combine exquisite cell-type selectivity with extraordinary potency.
Thus, there is a need to develop compositions and methods for treating central nervous system tumors including brain tumors utilizing antisense oligonucleotides. There is a need to develop compositions and treatment methods that result in down-regulation and inactivation of c-myb and other cellular products involved in uncontrolled cell proliferation of central nervous system tumor cells.